Project summary Neuronal nicotinic acetylcholine receptors (nAChRs) are implicated in several pathophysiological conditions including Alzheimer?s and Parkinson?s diseases, epilepsy, schizophrenia, and in the development of nicotine addiction. Therefore, pharmacological targeting of neuronal nAChR holds promise in the development of drug strategies to treat these conditions. The presence of multiple neuronal nAChR subtypes (e.g. ?4?2 and ?7 nAChR) with different subunit composition and unique physiological and pathological profiles hinders the development of nAChR selective therapeutics and only few neuronal nAChR drugs are clinically available. Since neuronal nAChRs share conserved ACh binding sites, it has proven difficult to develop agonists with high nAChR subtype selectivity. Moreover, direct activation of neuronal nAChRs by agonists is associated with alteration in cholinergic transmission due to prolonged activation and desensitization of nAChRs. As a result, positive allosteric modulators (PAMs) of nAChRs have emerged as a novel and more physiologically relevant strategy to enhance brain cholinergic transmission. PAMs do not bind to the ACh binding site or activate nAChRs in the absence of ACh. Rather, they potentiate ACh-induced responses by binding at site(s) distinct from the ACh binding sites. However, information regarding the location of nAChR PAM binding site(s) and the mechanisms responsible for PAM action are lacking. Our long-term goal is to determine the number and location of nAChR PAM binding sites and to develop nAChR subtype-selective PAMs for experimental and clinical uses. Our objective for this study is to identify binding sites for two structurally-unrelated ?4?2 nAChR PAMs (dFBr and CMPI). Our central hypothesis is that ?4?2 nAChRs have multiple PAM binding sites and that nAChR PAMs interact differently with these sites. We will us two complementary approaches: 1- Photoaffinity labeling with photoreactive nAChR PAMs to directly identify amino acids contributing to PAM binding sites; and 2- Mutational analyses coupled with in vitro electrophysiological recording to define the contribution of subunit interfaces in allosteric modulation of ?4?2 nAChR. The completion of this work will provide structural information that are important for understanding the diversity of allosteric binding sites in nAChRs and would facilitate the development of more selective ?4?2 nAChR PAMs.